Effects of calcium on stress and myosin phosphorylation in swine carotid media.

Phosphorylation of the 20,000 dalton myosin light chain is necessary for force generation, but not for force maintenance. The purpose of this study was to determine if the magnitude of the transient in myosin light chain phosphorylation affected force maintenance in swine carotid medial strips. Strips were either depolarized with K+ and then exposed to a Ca channel blocker (normal myosin light chain phosphorylation transient), or were exposed to the Ca channel blocker before K+ depolarization (blunted myosin light chain phosphorylation transient). The level of force maintained was the same regardless of the experimental protocol, indicating that the prior history of myosin light chain phosphorylation did not affect the maintained force. Whereas the phasic response to K+ depolarization of strips pretreated with Ca channel blocker was depressed in those treated with verapamil, only minimal effects were observed with nifedipine. These findings suggest an additional action for verapamil besides Ca channel blockade.

Ca2+, cAMP, and changes in myosin phosphorylation during contraction of smooth muscle.

Phosphorylation of myosin increases rapidly upon stimulation of an arterial smooth muscle. However, peak values are not maintained and phosphorylation declines, while active stress increases monotonically to a sustained steady state. The aim of this study was to determine the reason(s) for the transient change in myosin phosphorylation. Four hypotheses were considered: 1) reduced substrate, i.e., ATP depletion, 2) altered access of either the myosin kinase or phosphatase to the cross bridge, 3) reduced myosin kinase activity secondary to its phosphorylation by adenosine 3',5'-cyclic monophosphate-dependent protein kinase, and 4) reduced myoplasmic [Ca2+] during the contraction. Our results suggest that the most likely explanation is that there are two Ca2+-dependent regulatory processes: 1) myosin phosphorylation and 2) a second, unidentified site allowing stress maintenance with reduced cross-bridge cycling rates. A higher cell Ca2+ concentration appears to be necessary to activate myosin kinase and produce myosin phosphorylation than is needed for force maintenance. We suggest that agonist-induced Ca2+ transients, coupled with the differential Ca2+ sensitivity of the two regulatory systems, may explain the observed transient in myosin phosphorylation during a maintained contraction in smooth muscle.

Control of tone in vascular smooth muscle.

Total peripheral resistance and regional blood flow are determined by the contractile activity of vascular smooth muscle cells. The control systems that determine the activity of arterial smooth muscle are described. The interactions among control systems that underlie the remarkable diversity in the responses of blood vessels are emphasized to illustrate how various classes of drugs mediate clinically important effects.

Depression of contractions of rabbit aorta and guinea pig vena cava by mesudipine and other slow channel blockers.

The effects of several drugs having Ca++-antagonistic and vasodilating properties were compared in arterial and venous smooth muscles. Developed force (phasic component) was recorded from isolated rings (about 2 mm wide) of blood vessel wall taken from rabbit aorta or guinea pig inferior vena cava. The vascular smooth muscle (VSM) was stimulated to contract for a sustained period by elevating the extracellular K+ concentration ([K]o) to 100 mM or by exposure to norepinephrine (NE). All drugs depressed the K+-induced contractures in a dose-dependent manner between 10(-9) and 10(-5) M. The order of potency in aorta was: mesudipine = verapamil greater than diltiazem greater than nifedipine. Of the three drugs studied in vena cava, the order of potency was: mesudipine greater than verapamil greater than bepridil. It is concluded that, in both preparations of arterial and venous VSM, mesudipine is the most potent inhibitor of K+-induced contractions. Aortic contractions to 10(-7) M NE were depressed at concentrations of Ca++ antagonists 2 or 3 orders of magnitude less than those needed to depress contractions to 10(-5) M NE. The NE-induced contractions were depressed by the drugs to about the same extent as the K+-induced contractions when 10(-7) M NE was used, but were depressed to a much smaller extent when 10(-5) M NE was used. This may reflect the involvement of intracellular Ca++ storage sites in contractions induced by high NE concentrations. It is likely that these drugs depress and block Ca++ influx through the cell membrane.

Comparison of [3H]bepridil and [3H]verapamil uptake into rabbit aortic rings.

The efflux and uptake of [3H]Ca antagonists were studied in isolated rings of rabbit aorta. The efflux of [3H]bepridil occurred from two compartments. The faster (T1/2 = 0.6 min) initial compartment presumably represented efflux from the extracellular space. The slower (T1/2 = 25 min) component probably reflected efflux from the intracellular space. The uptake of [3H]bepridil into the cells occurred at an initial rate of 0.001 pmol/s . cm2, and by 80 min, uptake had reached steady state. Increasing the Ca2+ concentration to four times normal caused a marked decrease in the [3H]bepridil uptake, implying a possible competition between Ca2+ and bepridil. However, decreasing the Ca2+ concentration to one-fourth normal caused a slight decrease in [3H]bepridil uptake. [3H]Verapamil was not appreciably taken up by the tissues (less than 10% of [3H]bepridil uptake) unless the cell membranes were disrupted by digitonin treatment. (Lowering the Ca2+ concentration to one-fourth normal slightly increased. [3H]verapamil uptake.) These data suggest that bepridil can cross the sarcolemma (and therefore may exert some internal effect), whereas verapamil is effectively excluded from the cell interior by the cell membrane.

Bepridil (CERM-1978) blockade of action potentials in cultured rat aortic smooth muscle cells.

Reaggregate cultures (primary) were prepared from enzyme-dispersed vascular smooth muscle (VSM) cells from rat aortas. The cultures were incubated for 7-10 days, and then studied by the intracellular microelectrode technique. The cells were electrically quiescent (mean resting potential of --47 mV), and extracellular electrical stimulation usually did not elicit a membrane response. Addition of 10 mM tetraethylammonium rapidly induced excitability, allowing the VSM cells to fire Ca2+-dependent action potentials in response to electrical stimulation. The electrical responses often had two components, an initial spike and a later plateau-like component. The action potential spikes had a mean amplitude of 22 mV but occasionally were overshooting; the plateaus had a mean duration (at 50% repolarization) of 3.8 sec. A new anti-anginal agent, bepridil (10(-8)-10(-5) M), depressed the amplitude and duration of the plateau and blocked the spike component of the action potential in a dose-dependent fashion without affecting the resting potential. This finding is consistent with the view that bepridil acts as a Ca2+-antagonistic agent to prevent the generation of the action potentials, and this action can explain its antianginal properties.

Bepridil (CERM-1978) an verapamil depression of contractions of rabbit aortic rings.

Isolated rings (about 1 mm wide) of rabbit ascending aorta were stimulated to contract by norepinephrine (NE), increased extracellular potassium ion concentration, or electrical stimulation. When tested 20 min after addition, bepridil (CERM-1978) (10(-5)-10(-6) M), a new antianginal agent, and verapamil (10(-5)-10(-6) M) depressed the contractile responses to high K+ (30 mM) and NE (10(-6) M). Bepridil was almost as potent as verapamil in this action. Responses to strong electrical field stimulation were not affected by either agent. The depressed responses to NE in Ca-free EGTA-containing solutions were not further affected by bepridil or verapamil. Contractile responses to NE obtained from depolarized tissues, however, were markedly depressed by bepridil. These results suggest that bepridil, like verapamil, acts to inhibit contractions of vascular smooth muscle by decreasing influx of extracellular Ca++. In depolarized vascular smooth muscle, bepridil may also exert an effect to depress contractions supported by intracellular Ca++ release.